Optimization of separation for viscous suspensions

ABSTRACT

The present invention relates to methods and systems for optimization of dilution of a viscous starting material to isolate and/or concentrate the product of interest from the starting source material such that the process minimizes the volume of diluent and the total volume of the waste stream generated during the process as well as maximizing the yield of desired product. The system employs cross-flow filtration modules with sub-channels that are equidistant to the inlet and outlet of said modules and such modules are characterized by optimal channel height, optimal transmembrane pressure, etc., which are selected in order to achieve the best combination of product quality and production yield.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S. patentapplication Ser. No. 13/144,967 filed on Sep. 6, 2011 and now U.S. Pat.No. 8,795,530 which was filed under the provisions of 35 U.S.C. §371 andclaimed priority of International Patent Application No.PCT/US2010/021626 filed on Jan. 21, 2010, which in turn claimed priorityto U.S. Provisional Patent Application Ser. No. 61/146,142 filed on Jan.21, 2009 and U.S. Provisional Patent Application Ser. No. 61/148,959,filed on Jan. 31, 2009, the contents of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to separation of viscous source materials,and more particularly, to methods and systems for optimization ofdilution of a viscous starting material to isolate and/or concentratethe product of interest from the starting source material such that theprocess minimizes the volume of diluent and the total volume of thewaste stream generated as well as maximizing the yield of desiredproduct.

2. Discussion of Related Technology

Throughout the world more and more companies are looking to recovervalue added products from a wide variety of starting materials includingplants, roots, root crops, grains, flowers, animal tissue, cell culturescomprising yeast, algal, bacteria, or fungi species, milk, milkproducts, fruits and fruit juices. Additional companies are looking toextract value added products from solid and liquid waste streams such asmill and grain wash waters and fermentation bio-mass. One such wastestream will be the bio-mass from bio-fuel production which afterproduction of fuels such as diesel and alcohol will still be rich inplant proteins, sugars and carbohydrates. Another such waste stream willbe cellular bio-mass used for protein and essential fatty acidsproduction from wild and/or recombinant yeast, algae, bacteria, larvaeor fungi species.

A common practice for dry or solid starting materials is to solubilizestarting materials in a solvent such as aqueous and organic solvents sothat the valuable component becomes soluble in the solvent. The solutionis then processed by one or more of the known techniques of filtration,precipitation, extraction, chromatography and centrifugation to separatethe valuable components from the starting material and solvent. As aresult of growth in demand for naturally derived products companies areincreasing production of these products. Production costs andenvironmental issues such as the release of contaminated liquid wastestreams have pressured companies to extract more of the final productfrom the starting material and to minimize the use of solvents bypreparing larger more viscous process streams.

In recent years the science of cell culture has also endeavored toincrease production of cell derived products such as antibiotics,vaccine and therapeutic compounds by increasing the density of the cellcultures utilized to produce these highly valuable materials. Increasedcell density can be a highly beneficial as it allows for the increasedproduction of the final product in the same space as a less dense cellculture. It would seem that doubling the concentration of a cell cultureforming a viscous material should yield twice as much final productwithout any substantial increase in fermentation facility costs.

However, it has been found that all of these highly viscous materialsare far more difficult to process, such that, even though the cellculture is five (5) times denser the yield of final product is only 50%greater because the viscosity of the material prevents the separation ofthe desired target molecule from the mass of cellular materials. In thecase of extracts of solid phase material such as plants and animaltissue the problem is the same such that the viscous materials clogfilters and block chromatography columns as well as not separatingefficiently under normal centrifugal forces. One way of describing theproblem is to say that although larger crowds would contain more peopleable to buy a particular good or service it is harder to get the peoplewith money through the stores doors due to the congestion caused by thecrowd itself.

Although it would appear that a simple dilution of the viscous materialwould solve the problem, this creates at least four additionalproblems: 1) the cost of the diluent which can be highly expensive inthe case of diluents for pharmaceutical intended for human injection, 2)disposal of the higher volume of the waste stream, i.e. the originalvolume plus the volume of diluent, 3) the cost of the necessary tanksand mixing equipment in order to dilute the starting material, and 4)additional purification costs for the diluted final product.

As important as these problems are the single most important point is tohave the highest percentage of yield so that the initial purpose ofprocessing higher density materials is not negated by problems withrecovery of the desired product. Thus, it would be advantageous toprovide a method and system that provides higher yields from highdensity materials.

SUMMARY OF THE INVENTION

The present invention solves all of the aforementioned problems in asimple, inline, space efficient and continuous process that lowers costsand maximizes yield.

The present invention relates to the method and apparatus necessary todilute a viscous starting material to isolate and/or concentrate theproduct of interest from the starting material such that the processminimizes the volume of diluent and the total volume of the waste streamgenerated as well as maximizing the yield of desired product.

One such method employs one or more cross-flow filter units and theirassociated pumps, pipes and tanks It is also a further embodiment ofthis invention that the further purification of the target of interestcan be accomplished by complimentary purification techniques such aschromatography all as one unit of operation.

An extremely beneficial element of this method is that the process canbe readily modeled and optimized on the laboratory scale, with volumesas small as 0.5 L or in a continuous flow of 1 liter per minute forexample. This is extremely important in the pharmaceutical market aslarge volumes of highly specific therapeutic proteins are neitherinexpensive nor readily available. The separation methods of the presentinvention are envisioned in batch mode, continuous, or semi continuousmode.

One aspect of the present invention relates to a process for purifyingone or more target substances from a viscous source material, theprocess comprising:

-   -   contacting the viscous source material with a diluent in an        amount sufficient to reduce the viscosity of the viscous source        material and form a continuous stream of diluted source        material, wherein the diluent is contained in a separated vessel        from the viscous source material;    -   flowing the diluted source material into a recirculation loop of        a first cross-flow filter apparatus;    -   diafiltering the diluted source material with sufficient        diafiltration buffer so as to recover the desired yield of the        target substance by passing said target substance into the first        permeate fluid;    -   flowing the first permeate fluid containing the target substance        to a end product vessel;    -   flowing out the first retentate solution from the recirculating        liquid of the first cross-flow filter into a second cross-flow        filter unit, wherein the flow rate of the first retentate        solution is at the same flow rate as the diluted source material        being fed into the recirculation loop of the first cross-flow        filter apparatus;    -   diafiltering the flow of retentate into the second cross-flow        filter unit with sufficient diafiltration buffer so as to        recover the desired yield of the target substance by passing        said target substance into the second permeate fluid;    -   flowing the second permeate fluid containing the target        substance to the end product vessel;    -   concentrating the first and second retentate fluid by flowing        same to a third cross-flow filter apparatus communicatively        connected with the second cross-flow filter unit, wherein the        volume of the third retentate fluid is reduced to the        approximate volume of the undiluted source material or less        thereby forming a waste stream for further use;    -   recirculating the third permeate fluid back to the diluent        vessel for reuse;    -   concentrating the first and second permeate fluid by flowing        same to a fourth cross-flow filter apparatus communicatively        connected to the end product vessel wherein target substance is        concentrated and diafiltration buffer is removed in fourth        permeate stream and recirculated for reuse.

In another aspect, the present invention provides for a method of forseparating a target substance, the method comprising:

-   -   providing a diluent to a first reservoir;    -   providing a starting source material to a second reservoir:    -   providing a buffer to a third reservoir;    -   flowing a portion of the starting material with a portion of        diluent to form a mixture and flowing the mixture to a first        cross-flow filtration apparatus;    -   recirculating the mixture of diluent and starting material in        the first cross-flow filtration apparatus in a flow path adapted        for:        -   diafiltering the mixture;        -   permeating the target substance through the membrane;        -   selectively flowing a portion of the retentate of the first            cross-flow filtration apparatus to a second cross-flow            filtration apparatus;    -   recirculating the retentate of the first cross-flow filtration        apparatus across to a second cross-flow filtration apparatus in        a flow path adapted for:        -   selectively flowing a portion of the retentate out of the            second cross-flow filtration apparatus as a concentrate;        -   selectively flowing the permeate to a product reservoir; and    -   capturing the permeate of the first cross-flow filtration        apparatus in the product reservoir.    -   Optionally, the permeate fluid of the first cross-flow        filtration apparatus in the product reservoir can be        recirculated across a third cross-flow filtration apparatus in a        flow path adapted for:        -   concentrating the molecule of interest in the product            reservoir;        -   permeating the target substance free liquid into the third            reservoir;    -   selectively flowing the liquid in the third reservoir into the        first cross-flow filtration apparatus as the diafiltration        buffer.

In a still further aspect, the present invention provides for a systemcomprising:

a first reservoir constructed and arranged for holding a diluentsolution, and for selectively flowing liquid into and out of said firstreservoir;a second reservoir constructed and arranged for holding a startingmaterial, and for selectively flowing liquid into and out of said secondreservoir, the second reservoir can be the cell culture reservoir suchas a fermentor or culture bag;a first cross-flow filtration apparatus for separating liquids intopermeate and retentate streams, provided with means for flowing liquidin and permeate and retentate streams out of said first cross-flowfiltration apparatus;a second cross-flow filtration apparatus for separating liquids intopermeate and retentate streams, provided with means for flowing liquidin and permeate and retentate streams out of said second cross-flowfiltration apparatus;a third reservoir constructed and arranged for holding a buffer, and forselectively flowing liquid into and out of said third reservoir;a third cross-flow filtration apparatus for separating liquids intopermeate and retentate streams, provided with means for flowing liquidin and permeate and retentate streams out of said third cross-flowfiltration apparatus;a product reservoir constructed and arranged for holding the isolatedproduct, and for selectively flowing liquid into and out of said fourthreservoir; andconduit, valve and pump means constructed and arranged for:

-   -   providing an initial volume of diluent to the first reservoir;    -   providing an initial volume of buffer to the third reservoir;    -   selectively flowing a portion of the starting material with a        portion of diluent to form a mixture and flowing the mixture to        the first cross-flow filtration apparatus;    -   recirculating the mixture of diluent and starting material in        the first cross-flow filtration apparatus in a flow path adapted        for:        -   diafiltering the mixture;        -   permeating the target substance through the membrane;        -   selectively flowing a portion of the retentate of the first            cross-flow filtration apparatus to the second cross-flow            filtration apparatus;    -   recirculating the retentate of the first cross-flow filtration        apparatus across the second cross-flow filtration apparatus in a        flow path adapted for:        -   selectively flowing a portion of the retentate out of the            second cross-flow; filtration apparatus as a concentrate;        -   selectively flowing the permeate to the first reservoir;    -   capturing the permeate of the first cross-flow filtration        apparatus in the product reservoir and recirculating the        permeate fluid of the first cross-flow filtration apparatus in        the product reservoir across the third cross-flow filtration        apparatus in a flow path adapted for:        -   concentrating the molecule of interest in the product            reservoir;        -   permeating the target substance free liquid into the third            reservoir;    -   selectively flowing the liquid in the third reservoir into the        first cross-flow filtration apparatus as the diafiltration        buffer.

Yet another aspect of the invention provides for a process for isolationof a desirable product from a viscous starting mixture; the processcomprising the steps of:

-   -   diluting the starting mixture with a minimum amount of diluent        necessary for effecting passage of the target substance through        a first cross-flow filter membrane;    -   continually diafiltering the diluted material on the first        cross-flow filter membrane with sufficient diafiltration volumes        of buffer to achieve the desired yield of product in the        permeate; and    -   concentrating the permeate on a second cross-flow filter        membrane to recover the diluent for recycling while        simultaneously concentrating the permeate fluid containing the        product of interest on the second cross-flow filter membrane,        such that the product is concentrated.

Importantly, the product-free permeate is utilized and recycled as thediafiltration buffer such that at the end of the process, the producthas been isolated from the viscous starting mixture and concentratedinto a smaller volume, i.e. less than the volume of the undilutedstarting material. Further any remaining starting material is returnedto the initial undiluted viscous volume, or a lower volume, and nobuffers where consumed other than the initial volumes utilized to startthe process.

The present system and method may be carried out to effect a separationselected from the group consisting of: separating insect cell culturefluid into its constituent parts; separating viral culture fluid intoits constituent parts; separating an immunoglobulin from animmunoglobulin-containing culture of bacteria, yeast, algal, fungus,insect cells, or animal cells; separating an immunoglobulin from serum;separating a clotting factor from a clotting factor-containing cultureof bacteria, yeast, fungus, insect cells, or animal cells; separating aprotein from a protein-containing culture of bacteria, yeast, fungus,insect cells, or animal cells; separating an antigen from anantigen-containing culture of bacteria, yeast, fungus, insect cells, oranimal cells; separating an antigen from a viral culture containingsame; separating a hormone from a hormone-containing culture ofbacteria, yeast, fungus, insect cells, or animal cells; separatingessential fatty acids from a fatty acid containing culture of bacteria,yeast, algal, fungus, insect cells, larva or animal cells; separating aglycoprotein from a viral culture; and/or separating a glycoprotein froma glycoprotein-containing culture of bacteria, yeast, fungus, insectcells, or animal cells.

Other aspects and advantages of the invention will be more fullyapparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general scheme of an isolation system according to oneembodiment of the invention, using a cross-flow filtration basedapparatus

FIG. 2 A shows stacking of permeate and retentate sheets in thecross-flow filter of the present invention; B shows the sheet membersP/F/P according to another embodiment of the invention; and C shows planview of retentate sheet of the present invention

FIG. 3 shows the system components for diluting the source materialcomprising the desired product.

FIG. 4 shows the system components for moving the diluted sourcematerial through a cross-flow filtration stack and the addition ofbuffer to the cross-flow filtration wherein the permeate from the firstand second cross-flow filtration stacks is moved to a product reservoir.

FIG. 5 shows the system components for further concentration of thepermeate in the product reservoir wherein the buffer in the permeate isseparated and moved back to the buffer reservoir for reuse.

FIG. 6 show the recapturing of the diluent in the retentate and theseparation of waste cells.

FIG. 7 shows the full components of the system as described in FIGS. 3,4, 5 and 6.

FIG. 8 shows the system for passage of the product away from the dilutedcell suspension.

FIG. 9 shows the separation of product from the cell suspension byconstant diafiltration of the cells.

FIG. 10 shows the components required to concentrate diafilteredretentate of FIG. 8 to show the return of cell mass to original volumeof undiluted cell mass.

FIG. 11 shows the component necessary to separate the product from cellsby constant volume diafiltration while simultaneously concentrating theproduct.

DETAILED DESCRIPTION OF THE INVENTION

In the description of the present invention, certain terms are used asdefined below.

A “source material or starting material” as used herein refers to aviscous mixture containing solid and liquid materials such as mill andgrain wash waters, culture medium and fermentation bio-mass. The sourceor substance material are often complex mixtures or solutions containingmany biological molecules such as proteins, antibodies, essential fattyacids, hormones, and viruses as well as small molecules such as salts,sugars, lipids, etc. Examples of source or substance material that maycontain valuable biological substances amenable to the purificationmethod of the invention include, but are not limited to, a culturesupernatant from a bioreactor, a homogenized cell suspension, plasma,plasma fractions, milk, colostrum and cheese whey.

“Essential fatty acids (EFAs),” as used herein, means Omega-3 FattyAcids and Omega-6 Fatty Acid. EFAs are given the title ‘essential’ notonly because they are critical in promoting overall health, but becausethey cannot be manufactured by the body; therefore, it is essential thatintake is through diet. EFAs are considered to be long chainpolyunsaturated fatty acids (PUFAs). PUFAs of importance include, butare not limited to, docosahexaenoic acid (DHA), eicosapentaenoic acid(EPA), alpha-linolenic acid (ALA), gamma-linolenic acid (GLA),docosapentaenoic acid (DPA), arachidonic acid(all-cis-5,8,11,14-eicosatetraenoic acid; AA) and stearidonic acid(cis-6,9,12,15-octadecatetraenoic acid; SDA).

“Cross-flow filtration module” refers herein to a type of filter moduleor filter cassette that comprises a porous filter element across asurface of which the liquid medium to be filtered is flowed in atangential flow fashion, for permeation through the filter element ofselected component(s) of the liquid medium and include hollow fibers,spiral wound nodules, ceramic filters, cassette filters, plate and framefilters etc.

In a cross-flow filtration module employed in the present invention, theshear force exerted on the filter element (separation membrane surface)by the flow of the liquid medium serves to oppose accumulation of solidson the surface of the filter element. Useful cross-flow filters includemicrofiltration, ultrafiltration, nanofiltration and reverse osmosisfilter systems. The cross-flow filters can be used in parallel or seriesflow path stacked in a single housing or multi-element housing arrangedas a single or multiple loop system.

A preferred cross-flow filter system comprises a multiplicity of filtersheets (filtration membranes) in an operative stacked arrangement, e.g.,wherein filter sheets alternate with permeate and retentate sheets, andas a liquid to be filtered flows across the filter sheets, impermeate(non-permeating) species, e.g., solids or high-molecular-weight speciesof diameter larger than the filter sheet's pore size(s), are retainedand enter the retentate flow, and the liquid along with any permeatespecies diffuse through the filter sheet and enter the permeate flow(See FIG. 2 B). In a preferred embodiment of the present invention, suchcross-flow filtration module comprises a permeate collection anddischarge arrangement, a feed inlet, a retentate outlet, and multiplefluid-flow sub-channels that may for example be equidistant to the inletand the outlet as shown in FIGS. 2 A and C.

Cross-flow filtration modules and cross-flow filter cassettes useful inpractice of the present invention are commercially available fromSmartflow Technologies Inc, (Cary, N.C.), and are variously described inthe following United States patents: U.S. Pat. No. 4,867,876, “FilterPlate, Filter Plate Element, and Filter Comprising Same, issued Sep. 19,1989; U.S. Pat. No. 4,882,050, same title, issued Nov. 21, 1989; U.S.Pat. No. 5,034,124, same title, issued Sep. 11, 1990; U.S. Pat. No.5,049,268, same title, issued Sep. 17, 1991; U.S. Pat. No. 5,232,589,“Filter Element and Support, issued Aug. 3, 1993; U.S. Pat. No.5,342,517, “Filter Cassette Article,” issued Aug. 30, 1994; U.S. Pat.No. 5,593,580, same title, issued Jan. 14, 1997; and U.S. Pat. No.5,868,930, same title, issued Feb. 9, 1999; the disclosures of all ofwhich are hereby incorporated herein by reference in their respectiveentireties.

Briefly, a preferred cross-flow filter cassette of the present inventionis a stacked cassette filter assembly, as shown in FIG. 2A, in which thebase sequence of retentate sheet (R), filter sheet (F), permeate sheet(P), filter sheet (F), and retentate sheet (R) may be repeated in thesequence of sheets in the filter assembly as desired, e.g., in arepetitive sequence of retentate sheet (R), filter sheet (F), retentatesheet (R), filter sheet (F), permeate sheet (P), filter sheet (F),retentate sheet (R), filter sheet (F), permeate sheet (P), filter sheet(F), retentate sheet (R), filter sheet (F), retentate sheet (R). Thus,the filter cassette of a desired total mass transfer area is readilyformed from a stack of the repetitive sequences. In all repetitivesequences, except for a single unit sequence, the following relationshipis observed: where X is the number of filter sheets, 0.5X−1 is thenumber of interior retentate sheets, and 0.5X is the number of permeatesheets, with two outer retentate sheets being provided at the outerextremities of the stacked sheet array.

The filter sheets, and the retentate and permeate sheets employedtherewith, may be formed of any suitable materials of construction,including, for example, polymers, such as polypropylene, polyethylene,polysulfone, polyethersulfone, polyetherimide, polyimide,polyvinylchloride, polyester, etc.; nylon, silicone, urethane,regenerated cellulose, polycarbonate, cellulose acetate, cellulosetriacetate, cellulose nitrate, mixed esters of cellulose, etc.;ceramics, e.g., oxides of silicon, zirconium, and/or aluminum; metalssuch as stainless steel; polymeric fluorocarbons such aspolytetrafluoroethylene; and compatible alloys, mixtures and compositesof such materials.

Preferably, the filter sheets, retentate and permeate sheets are made ofmaterials which are adapted to accommodate high temperatures andchemical sterilants, so that the interior surfaces of the filter may besteam sterilized and/or chemically sanitized for regeneration and reuse,as “steam-in-place” and/or “sterilizable in situ” structures,respectively. Steam sterilization typically may be carried out attemperatures on the order of from about 121° C. to about 130° C., atsteam pressures of 15-30 psi, and at a sterilization exposure timetypically on the order of from about 15 minutes to about 2 hours, oreven longer. Alternatively, the entire cassette structure may be formedof materials which render the cassette article disposable in character.

In one particular aspect, the present invention relates to a filtrationcassette comprising a multilaminate array of sheet members of generallyrectangular and generally planar shape with main top and bottomsurfaces, wherein the sheet members include in sequence in the array afirst retentate sheet, a first filter sheet, a permeate sheet, andsecond filter sheet, and a second retentate sheet, wherein each of thesheet members in the array has at least one inlet basin opening at oneend thereof, and at least one outlet base opening at an opposite endthereof, with at least one permeate passage opening at longitudinal sidemargin portions of the sheet members;

-   -   each of the first and second retentate sheets having at least        one channel opening therein, wherein each channel opening        extends longitudinally between the inlet and outlet basin        openings of the sheets in the array and is open through the        entire thickness of the retentate sheet, and with each of the        first and second retentate sheets being bonded to an adjacent        filter sheet about peripheral and side portions thereof, with        their basin openings and permeate passage openings and register        with one another, and arranged to permit flow of filtrate        through the channel openings of the retentate sheet between the        inlet and outlet basin openings to permit permeate flow through        the filter sheet to the permeate sheet to the permeate passage        openings;    -   the filtration cassette comprising a unitary article of        inter-bonded sheet members.

In another embodiment, the present invention relates to a filtrationcassette comprising a multilaminate array of sheet members of generallyrectangular and generally planar shape with main top and bottomsurfaces, wherein the sheet members include in sequence in said array afirst retentate sheet, a first filter sheet, a permeate sheet, a secondfilter sheet, and a second retentate sheet, wherein each of the sheetmembers in said array has at least one inlet basin opening at one endthereof, and at least one outlet basin opening at an opposite endthereof, with at least one permeate passage opening at a longitudinalside margin portion of the sheet members;

-   -   each of the first and second retentate sheets having at least        one channel opening therein, extending longitudinally between        the inlet and outlet basin openings of the sheets in the array,        and being bonded (e.g., compression bonded) to an adjacent        filter sheet about peripheral end and side portions thereof,        with their basin openings and permeate passage openings in        register with one another and the filtrate passage openings of        each of the retentate sheets being circumscribingly bonded to        the adjacent filter sheet, and with a central portion of each of        the retentate sheets and adjacent filter sheets being unbonded        to permit permeate contacting the retentate sheet to flow        through the filter sheet to the permeate sheet; and    -   each of the filter sheets being secured at its peripheral        portions on a face thereof opposite the retentate sheet, to the        permeate sheet.

In yet another embodiment, the present invention relates to a filtrationcassette comprising a multilaminate array of sheet members of generallyrectangular and generally planar shape with main top and bottomsurfaces, wherein the sheet members include:

-   -   a first retentate sheet of suitable material, e.g. polysulfone,        polyethersulfone, polycarbonate, urethane, silicone, or other        material of construction, having (i) at least one longitudinally        extending rib or partition element, such partition element(s)        when provided in multiple configuration being transversely        spaced-apart from one another and being of substantially the        same height and substantially parallel to one another to define        a single or a series of channels between the partitions,        extending longitudinally between the respective inlet and outlet        basin openings of associated filter elements and permeate sheet        members, on both faces thereof, (ii) filtrate passage openings        at side portions of the sheets, and (iii) the retentate sheet        aligned to the first sheet of filter material at respective end        and side portions thereof, with the basin openings and filtrate        passage openings of the associated sheet members in register        with one another and the filtrate passage opening of the        retentate sheet member being circumscribingly compressed against        the first sheet of filter material, and with a central portion        of the first sheet of filter material and the retentate sheet        member being unbonded to permit permeate contacting the        retentate sheet member to flow through the first sheet member of        filter material to the permeate sheet member;    -   a first sheet member of filter material having (i) at least one        basin opening, of a suitable shape, e.g., polygonal,        semicircular, oval or sector shape, at each of opposite end        portions of the sheet member defining respective inlet and        outlet passages, and (ii) at least one filtrate passage opening        at the side portions of the sheet member, wherein the first        sheet member of filter material is bonded to the permeate sheet        member at their respective end and side portions, with their        basin openings and filtrate passage openings in register with        one another and the basin openings being circumscribingly bonded        at respective end portions of the first sheet member of filter        material and the permeate sheet member, and with a central        portion of the first sheet member of filter material and the        permeate sheet member being unbonded so as to define a central        portion permeate channel of the permeate sheet communicating        with the filtrate passages in the first sheet member of filter        material and in the permeate sheet member;    -   a permeate sheet member, having (i) multiple basin openings of        suitable shape at each of opposite end portions of the sheet        member defining respective inlet and outlet passages, and (ii)        filtrate passage openings at the side portions of the sheet        member;    -   a second sheet member of filter material having (i) at least one        basin opening at each of opposite end portions of the sheet        member defining respective inlet and outlet passages, and (ii)        at least one filtrate passage opening at the side portions of        the sheet member, wherein the second sheet member of filter        material is compression sealed to the retentate sheet member at        their respective end and side portions, with their basin        openings and filtrate passage openings in register with one        another and the filtrate passage opening of the retentate sheet        member being compression sealed to the second sheet member of        filter material, and with a central portion of the second sheet        member of filter material and the retentate sheet member being        unbonded to permit permeate contacting the retentate sheet        member to flow through the second sheet member of filter        material; and    -   a second retentate sheet member of suitable material, e.g.        polysulfone, polyethersulfone, polycarbonate, urethane,        silicone, having (i) at least one longitudinally extending rib        or partition element, provided that when multiple partition        elements are employed, the partition elements are transversely        spaced-apart from one another, such partition elements being of        substantially the same height and substantially parallel to one        another, to define a single channel or a series of channels        between the partitions, extending longitudinally between the        respective inlet and outlet basin openings of the filter        elements and permeate sheet members, on both faces thereof, (ii)        filtrate passage openings at the side portions of the sheet        member, and (iii) the retentate sheet compression sealed to the        second sheet of filter material at respective end and side        portions thereof, with their basin openings and filtrate passage        openings in register with one another and the filtrate passage        opening of the retentate sheet member being compression sealed        to the second sheet member of filter material, and with a        central portion of the first sheet member of filter material and        the retentate sheet member being unbonded to permit permeate        contacting the retentate sheet member to flow through the second        sheet member of filter material to the permeate sheet member.

The end plates used with the cassette articles of the invention to forma unitary filter assembly may be formed of any suitable materials ofconstruction, including, for example, stainless steel or other suitablemetal, or polymers such as polypropylene, polysulfone, andpolyetherimide.

Specifically, the present invention employs cross-flow filtrationmodules with sub-channels that are equidistant to the inlet and outletof said modules such as shown in FIGS. 2A and 2C (retentate sheet).Moreover, said cross-flow filtration modules are characterized byoptimal channel height, optimal transmembrane pressure, optimal membranepore size and pore structure, optimal membrane chemistry, etc., whichare selected in order to achieve the best combination of product qualityand production yield.

For example, shear at the surface of the membrane is critical inminimizing gel layer formation, but excessive shear is deleterious inthe following three key aspects: (1) excessive shear increases energyconsumption, (2) excess shear interferes with diffusion at the membranesurface, upon which separation process directly depends, (3) excessiveshear can deprive certain compounds of their bioactivities. It istherefore desirable to maintain shear within an optimal range.

Furthermore, it is possible to optimize the separate processes withcross-flow filtration modules of variable channel velocities but ofuniform channel heights, given the fact that most commercial cross-flowmodules are only available in a single channel height. When the channelheight of a cross-flow filtration module is known, shear is directlyproportional to channel velocity of such module for the same solutionpassing by.

In the literature, numerous techniques have been proposed to effect theseparation of target substances using membrane separations with additionof foreign substances such as acid, base, salt and solvents. In contrastto these chemical additives-based methods, the methodology of thepresent invention permits a target substance to be separated from aninput fluid by the simplest mechanical means. In the use of cross-flowfiltration modules of the type described in the aforementioned patents,the specificity and speed of a desired separation is effected by a)fluid distribution in the cross-flow module, b) channel height of thecross flow module, c) channel length, d) shear rate, e) membrane porestructure, f) membrane structure, g) membrane chemistry, h)trans-membrane pressure, and i) pressure drop, which is a function ofchannel length, velocity and solution viscosity.

The approaches by others involving various additives and manipulationsof transmembrane pressure appear to be predicated on overcoming problemscreated by poor distribution of flow within the cross-flow module. It isnot to say that the addition of salts and solvents do not have a placein separation but without proper flow distribution the membraneseparation cannot be optimally operated nor will cleaning techniques befully beneficial. It will be appreciated, based on the disclosure hereinthat numerous heretofore expensive or difficult separations are renderedfar simpler and more economical by employing the techniques describedherein.

Thus, the invention relates in another aspect to optimizing the membraneseparation process, comprising:

-   -   selecting a cross-flow membrane module wherein the distance from        the inlet port to the outlet port is equidistant from the inlet        to outlet for each sub-channel of the device, i.e., each        sub-channel is of a same dimensional character;    -   selecting an optimal channel height;    -   selecting an optimal shear rate and/or channel velocity;    -   selecting an optimal transmembrane pressure;    -   selecting an optimal membrane pore size;    -   selecting an optimal temperature;    -   selecting an optimal channel length; and    -   selecting an optimal pressure drop which is the composite of        -   the optimal channel height;        -   the optimal shear rate and/or channel velocity;        -   optimal channel length; and        -   the viscosity of the solution being filtered.

Selecting a channel height can be performed mathematically orempirically by trial and error. In most cell fermentation applications,trial and error has been more appropriate due to the fact that theviscosity of the cell broth or product solution is rarely known, thecell count and cell viability are highly variable, and the solution isfrequently non-Newtowian. The objective of channel selection is tominimize channel height with three critical stipulations: first, thechannel must be sufficiently high to allow the unrestricted passage ofany larger material such as clumped cells; second, the channel shouldnot cause excessive pressure drop and loss of linear efficiency; andthird, the channel should be sufficiently high as to allow the properangle of attack for substances to encounter the membrane pore and passthrough the pore. The optimal channel height is dependent on the lengthand viscosity of the solution.

Several notable observations have been made in initial trials andprocess scale-up, as discussed below.

For cell suspensions having an optical density (OD) of 2 to 500, and apath length of 6 to 12 inches, start with a channel height between 0.4to 0.75 mm. If the inlet pressure is above 15 PSIG at a velocity of 2.0M/sec, then the channel is too thin.

For cell suspensions having an optical density (OD) of 2 to 500, and apath length of 6 to 12 inches, start with a channel height between 0.4to 0.75 mm. If the inlet pressure is below 5 PSIG at a velocity of 2.0M/sec the channel is too high.

For cell suspensions having an optical density (OD) of 2 to 500, and apath length of 25 to 40 inches, start with a channel height between 0.7to 1.0 mm. If the inlet pressure is above 15 PSIG at a velocity of 2.0M/sec, the channel is too thin.

For cell suspensions having an optical density (OD) of 2 to 500, and apath length of 25 to 40 inches, start with a channel height between 0.7to 1.0 mm. If the inlet pressure is below 5 PSIG at a velocity of 2.0M/sec, the channel is too high.

Shear at the surface of the membrane is critical in minimizing gel layerformation, but excess shear is deleterious in at least three keyaspects: first, it increases energy consumption costs; second, excessshear and the resulting pressure has been demonstrated to interfere withseparations which appear to be based on diffusion at the membranesurface; and third, shear can result in damage to cells and impairmentof the bioactivity of certain compounds. It is apparent that thebenefits of shear are readily observed within a specific range for eachprocess and that shear rates outside that range are highly destructive.

Before progressing in the explication of the optimization process, itmust be pointed out that the shear stability of the substances insolution or suspension is a key element in shear optimization. Onlythrough accurately calculating and charting the specific shear ratesutilized during optimization can the true benefits of shear optimizationbecome apparent. In concentration processes, it is graphically clearthat the higher the shear, the higher the membrane flux, with twostriking observations.

First, there is a minimum shear value that minimizes the gel-layerformation. This minimum shear can be best demonstrated for any specificsolution by first running the device at an excessively high shear rateand then systematically lowering the shear incrementally until theresultant flux decay of each incremental reduction in shear isdisproportional to the reduction in shear. Given the repeatedobservation during cross-flow concentration applications that increasingthe shear increases the flux, the maximum flux for solutions is clearlygoverned by the law of diminishing returns, where at some pointincreases in shear provide lower increases in flux.

For concentration applications, it can be stated that there is a minimumshear required to keep the membrane clean through minimizing thegel-layer formation, as well as a maximum shear which is determined bythe cost of energy required to marginally increase flux.

For separation applications it can be stated that there is a minimumshear required to minimize the gel-layer formation and allow the passageof a target substance, as well as a maximum shear that interferes withthe passage of a target substance, even though the higher shear resultsin higher water flux rates.

Furthermore, it is possible to develop processes based on channelvelocity, given that most cross-flow end users tend to work with asingle channel height based on past experiences, and the predominance ofcross-flow modules that are only available in a single channel height.

When working with a single device of uniform height, shear is directlyproportional to channel velocity for the same solution. In concentrationapplications, the end user should install a flow meter on the permeateport and record the maximum flux obtained at reasonable cross-flowvelocities between 1 and 4 M/sec for devices with channel heightsbetween 0.5 mm and 1.0 mm. In separation applications, the end usershould assay the passage of the target material(s) at cross-flowvelocities between 0.5 and 2.5 M/sec for devices with channel heightsbetween 0.5 mm and 1.5 mm

The optimization of transmembrane pressure (TMP) can only be performedafter the appropriate tangential velocity has been determined.Transmembrane pressure is calculated as TMP=(inlet pressure+outletpressure)/2−permeate pressure. It is imperative that the tangentialvelocity (flow rate) be monitored during the optimization oftransmembrane pressure, since increasing the pressure normally decreasesthe output of most pumps due to slippage. The objective of theoptimization of transmembrane pressure is to define the correlation oftransmembrane pressure to permeate flow rate. The normal relationship isa traditional bell curve. A graph of transmembrane pressure versuspermeate flow rate should resemble a bell curve. Increases intransmembrane pressure cause increases in the permeate rate until amaximum is reached, and thereafter further increases in transmembranepressure result in decreases in the permeate rate. The reason for thisresult is that the decreasing flow rate, resulting from highertransmembrane pressures, is the result of gel layer and/or membranecompression.

The procedure is set out below:

-   -   (1) Operate the system in total recycle mode at the optimum        tangential velocity for sufficient time, typically fifteen        minutes, for any gel layer to accumulate.    -   (2) Measure the permeate rate. This is the Base Rate.    -   (3) Increase the transmembrane pressure by 3 PSIG and measure        the permeate rate immediately and after five minutes at the new        transmembrane pressure. Compare the permeate rates to the base        rate. If the rates have increased go to Step 4. If the rate        decreases go to step 5.    -   (4) Repeat steps 2 and 3 until the permeate rate no longer        increases during each step or does not hold that increase for        five minutes.    -   (5) The optimum transmembrane pressure is the last pressure        reading where the increase in pressure result in an increase in        permeate rate.

In separation applications, the end user should assay the passage of thetarget material(s) at TMP's between 2 and 15 PSIG where the cross-flowvelocity is optimized between 0.5 and 2.5 M/sec for devices with channelheights between 0.5 mm and 1.5 mm.

Selecting and optimizing the channel length has been totally impracticalif not an impossible task until the advent of the stacked cross-flowfiltration units as described herein. The inherent difficulty ofoptimizing the channel length in prior art devices has been three-fold:first, the devices such as spirals were designed to maximize membraneutilization based on the width that membranes could be cast rather thanany other factor; second, increases in channel length for devices suchas cassettes resulted in enormous increases in pressure drop due to thepredetermined channel geometry imposed by the retentate screen; andthird, plate and frame devices, such as for example Pleidae by Rhodia,France, use fixed molded plates which are manufactured in a singlelength and cannot be changed without manufacturing a new mold.

The present invention eliminates these prior art restrictions byproviding the ability to select the channel length by utilization of aninfinitely variable retentate sheet that is cut to length from anappropriately manufactured film, selected from a variety of standard orstarting point thicknesses. Likewise, the membrane sheets and permeatesheets are cut to matching lengths and laminated into a stackedcassette.

There undoubtedly are many ways of selecting the optimum membrane forany given process, yet it appears the most reliable method of usingmembranes is to consider the manufacturer's specified pore size as atheoretical starting point which then is modified by the solution andthe operating conditions. As a result of numerous trials, a practicalparameter has been determined and termed the coefficient of rejection.

Coefficient of Rejection (CRV)

Membranes have a rejection characteristic (value) that is first orderand is defined by the size, charge and shape of the pore. For simplicitythe CRV, coefficient of rejection value, is the stated pore sizeprovided by the manufacturer. In purifying a product of interest the CRVof a membrane is more important for separation applications as comparedto concentration applications. The rules below specifically relate toseparation applications. These effects will vary in concentrationapplications.

The CRV of a membrane is subject to the velocity of the tangential flowoperation. Empirical evidence suggests that the neutral point of anymembrane can occur in two zones, the first zone being the point at whichthe transmembrane pressure and/or shear compress the gel layer and theCRV increases, and the second zone occurring where the TMP and velocityminimize the shear and the CRV decreases. The neutral point (NP) isdefined as the point where a membrane freely passes particles 0.5 timesthe stated pore size, NP=0.5(Pore Size).

Therefore:

-   -   the effective CRV of a typical micro porous membrane is greater        than the pore size, for velocities greater than 1.5 M/sec and        less than 3.0 M/sec.; and    -   the effective CRV of a typical ultrafiltration membrane is        greater than the pore size, for velocities greater than 1.5 and        less than 3.0 M/sec.

Example

A 0.3μ particle may freely pass a 0.45μ polymeric membrane when the

velocity is between 1.5 and 4.0 M/sec but not for velocities between 0.5and 1.5 M/sec or 4.5 and 12 M/sec.

Example

A 45,000 MW protein may freely pass a 0.2μ membrane for velocities of 0to 1.0 M/sec but be significantly retained when the velocity isincreased above 1.5 M/sec. In the same experiment, it was documentedthat protein passage was above 90% for velocities between 0.8 and 1.5M/sec and 25% for a velocity of 2.0 M/sec. Additionally, this sameprotein had 65% membrane transmission through a 100,000 MW membrane atvelocity of 1.0 M/sec.

Further,

-   -   the CRV of a membrane is proportional to the molarity of the        solution;    -   the greater the solute concentration, the greater the CRV; and    -   the lower the solute concentration, the smaller the CRV.

Thus, a membrane may have a stated pore size of 500,000 MW but willretain proteins of 110,000 MW in cell suspension with an OD over 100 andpass the same 110,000 MW protein when the OD is less than 50.

The process can be developed and optimized by empirical testing ofundiluted and/or diluted volumes of starting source material to measurethe percent of target molecule passed into the permeate fluid. Twotesting methodologies can be employed including:

-   -   1) Concentrate the undiluted and/or diluted material as much as        possible, from 1 to 10X for example, collect and assay samples        of the retentate fluid and the permeate fluid simultaneously        collected at various points in the concentration process such as        start, 2X, 3X, 5X, 7X and 10X, divide the assayed level of        target substance measured in the permeate fluid by the assayed        level of target substance in the retentate sample that was taken        at the same point in time and multiply by 100 in order to        express the result as percent passage of the target material.    -   2) Continuously diafiltering the undiluted and/or diluted        material against multiple volumes, from 1 to 10X for example,        collect and assay samples of the retentate fluid and the        permeate fluid simultaneously collected at various points in the        diafiltration process such as start, 2X, 3X, 5X, 7X and 10X,        divide the assayed level of target substance measured in the        permeate fluid by the assayed level of target substance in the        retentate sample that was taken at the same point in time and        multiply by 100 in order to express the result as percent        passage of the target material.

The data from these two processes will indicate several key factorswhich will provide a total isolation process as described herein:

-   -   a) The appropriate dilution of the starting material that        results in good passage of the desired product away from the        starting material.    -   b) The number of diafiltration volumes necessary to achieve an        acceptable yield.    -   c) The degree of concentration to which the starting material        can be concentrated.    -   d) The membrane performance of the tested membranes at the        operating conditions utilized in the testing.    -   e) Optimization of the membrane performance.

A succinct description of the process would be to start the isolation ofa desirable product from a viscous mixture by diluting the startingmixture the minimum amount necessary to effect good passage of thetarget substance through a separating membrane, followed by continuallydiafiltering the diluted material on said separating membrane withsufficient diafiltration volumes to achieve the desired yield, then toconcentrate the diluted mixture on a second membrane device to recoverthe diluent for recycling while simultaneously concentrating thepermeate fluid, containing the product of interest that was in themixture, on the separating membrane, such that the product isconcentrated. Then the product-free permeate is utilized and recycled asthe diafiltration buffer such that at the end of the process, theproduct has been isolated from the viscous mixture and concentrated intoa smaller volume, i.e. less than the volume of the undiluted startingmaterial. Further any remaining starting material is returned to theinitial undiluted viscous volume, or a lower volume, and no bufferswhere consumed other than the initial volumes utilized to start theprocess.

Another way to understand the invention is to look at how the fluidflows through the various steps mathematically:

The terminal retentate flow (TRF), in liters per hour, for the startingmaterial concentration step (SMCS) is approximately equal to thestarting volume (SV) of the starting material, in liters, divided by thedesired processing time (DPT), hours. TRF (LPH)=SV (L)/DPT (h)

In preferred embodiments of the apparatus, the terminal retentate flowderived from the starting material concentration step (SMCS) can bechanged to a fraction of the starting volume (SV) flow rate bydecreasing the volume of starting material in order to lower the wastestream or to concentrate the remaining dry matter in the starting volume(SV) as this fluid stream may be a valuable by-product. One such examplewould be to utilize the invention to isolate one or more proteins and/orcarbohydrates from a plant material such as soy, potato, tobacco andmilk where the starting material less the protein or carbohydrate hadresidual value as a bulk protein or additive to a third product such assoy flour, milk powder, and fish feeds etc. The reduced volume wouldlower the cost of either drying or transporting the liquid stream.

The feed flow rate (FF), in liters per hour, into the separating filterapparatus (SFA) is equal to the terminal retentate flow rate (TRF) whereno concentration of the starting volume is desired. FF (LPH)=TRF(LPH)=SV (L)/DPT (h)

If for example the terminal retentate flow rate (TRF), in liters perhour, is to be one-half (½) of the starting solution flow rate (PF) whenthere is no dilution of the starting solution, then the equation issimply modified. FF (LPH)=PF (LPH)=2xRF (LPH)=0.5x(SV (L)/DPT (h))

Further, the feed flow rate (FF), in liters per hour, into theseparating filter apparatus (SFA) is equal to the sum of the dilutingfluid flow rate (DF) plus the product flow rate (PF). FF (LPH)=DF(LPH)+PF (LPH)

The retentate fluid flow (RFF), liters per hour, from the separatingfilter apparatus (SFA) into the starting material concentration step(SMCS) is equal to the feed flow rate (FF) when the feed flow rate isneither diluted or concentrated by the separating filter apparatus(SFA).

The diluting fluid flow rate (DF) is equal to the desired initialdilution for the product flow rate. If for example, it was determinedthat the product of interest could be separated when the startingmaterial was diluted with an equal volume of buffer than the equationwould be DF=PF wherein we could say that FF (LPH)=2x(SV (L)/DPT (h)).

If for example, it was determined that the product of interest could beseparated when the starting material was diluted with two equal volumesof buffer than the equation would be DF=2xPF wherein FF=3xPF=3x(SV/DPT).

If for example, it was determined that the product of interest could beseparated when the starting material was first concentrated in halfbefore entering the separating filter apparatus (SFA) than the equationwould be FF=0.5xPF.

If for example, the starting volume (SV) was to be concentrated in halfwithin the separating filter apparatus (SFA) before initiating thediafiltration fluid flow than the equation would be TRF=0.5xPF.

The permeate fluid from the starting material concentration apparatus(PCA) replaces the diluting fluid flow rate (DF) that is utilized todilute the starting volume as necessary. The equation for thisrelationship is PCA=DF.

The flow rate of diafiltration buffer (DFB) into the separating filterapparatus (SFA) is determined by the number of diafiltration volumesnecessary to pass the target molecule into the permeate stream of theseparating filter apparatus (SFA) in order to recover the desired yieldof the target molecule. In the case where the diluting flow rate,expressed as DF, resulted in a process where the target substance passedfreely into the permeate stream than the following table can be utilizedto determine the yield of the target substance based on the number ofdiafiltration volumes.

Solute Recovery vs. Volume Replacement Recovery of Target Molecule inthe Permeate Fluid i.e. when passage is Volume Replacement unrestricted,0% rejection 0 0 1   50% 2   75% 3 87.5% 5 96.9% 7 98.7% 10 99.8%

In the case where the feed flow rate (FF) is to undergo diafiltrationwithout being concentrated or diluted in the separating filter apparatus(SFA) than the feed flow rate will equal the retentate fluid flow (RFF)to the starting material concentration step (SMCS) and the permeate rate(PSA) of the separating filter apparatus is equal to the flow rate ofdiafiltration buffer (DFB) into the separating filter apparatus (SFA).

-   -   If for example, the process is determined to require a five (5)        fold diafiltration of the feed flow rate (FF) in order to obtain        a yield of 96.9%, as shown in the table, than the equation can        be expressed as DFB=5xFF.

The permeate flow rate of the product concentration apparatus (PCA)needs to replace the permeate fluid discharged from the separatingfilter apparatus (SFA) as permeate flow rate of the separating filterapparatus is equal to the diafiltration buffer (DFB) flow rate such thatthe equation is PCA=DFB.

In preferred embodiments of the apparatus, it maybe advantageous tointermittently harvest the concentrated product from the productreservoir to avoid prolonged exposure to the shear forces of theconcentrating membrane apparatus or simply to avoid product degradationover time as a result of varied biological and/or chemical effects.

FIG. 1 shows an arrangement of reservoirs and cross-flow filtrationunits that is representative of one embodiment, understanding that asystem may include from one to multiple reservoirs and cross-flowfiltration units, the present system comprising:

A first reservoir 1 constructed and arranged for holding a diluentsolution, and for selectively flowing liquid into and out of said firstreservoir;

A second reservoir 2 constructed and arranged for holding a viscousstarting source material, and for selectively flowing liquid into andout of said second reservoir, the second reservoir preferably is a cellculture reservoir such as a fermentor or culture bag; wherein the firstand second reservoir are communicatively connected to a channel 3 fordelivering components of the first and second reservoir and combiningtherein for delivery to at least one cross-flow filtration unitpositioned downstream of the combining channel;

A first cross-flow filtration apparatus 4 for separating liquids intopermeate and retentate streams, provided with means for flowing liquidin and permeate and retentate streams out of said first cross-flowfiltration apparatus, wherein the permeate includes at least the targetof choice and can be directed to a end product reservoir 6 and whereinthe retentate comprises cell mass and/or culture material for movementdownstream or recirculation into the first cross-flow filtration unit;

A second cross-flow filtration apparatus 5 communicatively connected tothe first cross-flow filtration unit and retentate stream leavingtherefrom, wherein the second cross-flow filtration unit is used forseparating the retentate stream into permeate and retentate streams andprovided with means for flowing liquid in and permeate and retentatestreams out of said second cross-flow filtration apparatus, wherein thepermeate includes at least the target of choice and can be directed tothe end product reservoir 6 and wherein the retentate comprises cellmass and/or culture material for movement downstream or recirculationinto the second cross-flow filtration unit;

A third reservoir 7 constructed and arranged for holding a diafiltrationbuffer, and for selectively flowing liquid into and out of said thirdreservoir; wherein the buffer is deliverable, though a channel system10, to the first and second cross-flow filtration units and for mixingwith the input stream therein;

A third cross-flow filtration apparatus 8 for separating retentatestream from the second cross-flow filtration apparatus into permeate andretentate streams, provided with means for flowing liquid in andpermeate and retentate streams out of said third cross-flow filtrationapparatus, wherein the third cross-flow filtration unit iscommunicatively connected to the retentate stream of the first and/orsecond cross-flow filtration unit; wherein the dilution buffer isremoved via the permeate stream for optional recirculation into dilutionbuffer reservoir 1 and the retentate stream which includes waste cellscan be optionally used for multiple purposes including furtherseparation of additional target molecules or used in feed products foranimals, both terrestrial and aquatic.

The end product reservoir 6 constructed and arranged for holding theisolated end product, and for selectively flowing liquid into and out ofsaid end product reservoir; wherein the end product is removed directlyfrom the end product reservoir or in the alternative directed through aseparation cross-flow filtration unit 9 for separation of end productfrom at least the diafiltration buffering solution, wherein thediafiltration buffering solution can be directed to the buffer reservoir7 for reuse in the system.

The system further comprises conduit, valve and pump means constructedand arranged to move liquid and slurries from different reservoirs tocross-flow filters. In preferred embodiments of the apparatus, thereservoirs are provided with thermal jackets to maintain appropriateprocess temperatures.

An illustrative example is provided using the system of OPTISEP®filtration modules for processing Pichia pastoris. The present examplecan be used to separate expressed proteins from high cell density P.pastoris cell culture, wherein the starting concentration of 50% solidsis able to provide a recovery of 95%+.

The process comprises diluting the Pichia so that it is readily filtered(step 1), then filtering the diluted material in a first OPTISEP® filtermodule via diafiltration so as to separate the product from the feedstock (step 2), while simultaneously: a) concentrating the permeate on asecond OPTISEP® filter module which both concentrates the product andrecycles the diafiltration buffer (step 3) and b) concentrating theretentate of the first OPTISEP® filter module with a third OPTISEP®filter module recovering the diluent and returning the feed stock to itsoriginal volume or less (step 4).

Typical P. pastoris fermentations can reach a wet cell weight of 50 to60%. At these high solid concentrations, the culture typically must bediluted to permit effective passage during filtration. The dilution step1 is depicted in FIG. 3 and includes the following observations and orparameters:

-   -   The cell culture is diluted to a predetermined concentration        with diluent.    -   The flow rate of cell culture fluid into the diluent is equal to        the volume of cell culture fluid divided by the desired        processing time.    -   Increasing the amount of diluent increases the effective        separation of product from the cell suspension.    -   Increasing the amount of diluent increases the flux rate of the        membrane.    -   Increasing the amount of diluent decreases the operating        pressure.    -   Increasing the amount of diluent increases the total volume of        liquid to be processed.

Step 2, once the culture is diluted; the cells are separated from theproduct in solution using an OPTISEP filter module with amicrofiltration (MF) membrane. In this process, the product passesthrough the MF membrane into the MF permeate by continuousdiafiltration, as labeled in step 2 (FIG. 4) the cells remain in therecirculation loop, i.e. the retentate fluid. The MF membrane capacityis increased by adding more membrane area to the filter holder and/ormore recirculation loops. The attached depiction of FIG. 4 shows two (2)recirculation loops in series with one filter holder in each loop.Because the diafiltration is a steady state diafiltration, the volumeentering the loop (i.e. the feed rate) is equal to the volume leavingthe loop (i.e. the bleed rate) and the volume of permeate leaving theloop (i.e. permeate rate) is equal to the volume of diafiltration bufferentering the system (i.e. the diafiltration rate.) Therefore, theconcentrations of the cells entering, leaving, and inside therecirculation loop are constant at the optimal concentration set in thedilution step (step 1). The concentrations of the molecules that passthrough the membrane such as the product are reduced. The source of thediafiltration buffer is described in the third step. The concentrationstep 2 includes the following observations and/or parameters:

-   -   The flow rate into the MF stage is equal the flow rate out of        the MF stage    -   The permeate rate out of the MF stage equals the flow rate of        diafiltration buffer into the MF stage.    -   Increasing the diafiltration factor will increase the product        yield.

The third step (Step 3, FIG. 5) is the concentration of the productderived from the MF permeate fluid as well as generating thediafiltration buffer. Utilizing a second OPTISEP filter modulecontaining an ultrafiltration (UF) membrane, the permeate fluid of theMF membrane containing the product protein is concentrated. The productis concentrated in the retentate of this filter as depicted in FIG. 5.The UF permeate is recycled back to step 2 as the diafiltration buffer.This recycling dramatically lowers the waste produced from the systemand decreases the operating expenses through the virtual elimination ofbuffers normally required for diafiltration. The concentration of theproduct includes the observations and/or parameters:

-   -   MF permeate containing the product is concentrated.    -   The rate of concentration is equal to the rate of the        diafiltration of step 2.    -   Product can be continually harvested from the product retention        loop or the product vessel if desired.

The fourth and final step is the concentration of the cells back totheir original concentration or a higher concentration using the thirdOPTISEP filter module with a UF membrane, step 4 (FIG. 6). Byconcentrating the cells, the volume of cell waste is decreased. Therecycle of the permeate dramatically lowers the waste produced from thesystem and decreases the operating expenses through the virtualelimination of the diluent needed to lower the concentration of theoriginal fermentation broth. In certain situations this step could beaccomplished with an MF filter such that the number of diafiltrationrequired in step 2 would be reduced and the permeate flow paths would bealtered slightly from the attached slides. The concentration of thecells back to the original concentration includes the followingobservations and/or parameters:

-   -   The diluted cell broth is concentrated back to the original cell        concentration or greater.    -   The permeate fluid of the concentration is recycled back to be        reused as a diluent.    -   The final volume of cell paste can be less than the volume of        the fermentor.    -   The final volume of cell paste can be chemically and/or heat        treated in line for discharge.

One advantage of separating the overall process into these 4 distinctunit operations is that each unit operation can be studied, understood,and optimized independently. Then the optimized parameters can beimplemented when designing the large scale design.

FIG. 7 shows the entire process without the various visual keys.

Optimization of the process includes the following experiments asoutlined in FIGS. 8, 9, 10 and 11 including;

Experiment 1

The purpose of experiment 1 is to demonstrate the passage of the productaway from the diluted cell suspension. Experiment 1 is performed atvarious dilution rates to optimize product recovery, minimize the rateof dilution and maximize the membrane performance in liters per metersquare per hour (LMH). The starting material is diluted in a batch modeusing different levels of diluent to determine the appropriate dilutionand the product is separated from the diluted starting material viaconstant volume diafiltration. The level of diafiltration is determined.

Experiment 2

The purpose of experiments 2 is to concentrate the permeate ofexperiment 1. The permeate of experiment 1 contains the product whichwas separated from the cells by constant volume diafiltration of thecells. The permeate of the separation is concentrated in order todemonstrate recovery of the product and number of passes through MF foracceptable product concentration.

Experiment 3

The purpose of experiments 3 is to concentrate the diafiltered retentateof experiment 1 in order to demonstrate the ability to return the cellmass to the original undiluted volume or less. In other words thepurpose of experiment 3 is to show the feasibility of reducing thevolume of the process waste stream as well as the ability to recover thediluent. The diluted cellular material is concentrated to the originalstarting volume or less and number of passes through MF for acceptableconcentration.

Another embodiment for optimization comprises performing experiments 1,2 and 3 utilizing three (3) different filtration steps (MF, UF of the MFpermeate, and UF of cells); followed by a experiment 4 (FIG. 11) whichis the simultaneous operation of the MF separation and the UFconcentration of the MF permeate fluid

Experiment 4

The purpose of Experiment 4 is the separation of the product from thecells by constant volume diafiltration while simultaneouslyconcentrating the product such that the product is concentrated and thepermeate of the product concentration is recycled as the diafiltrationbuffer. The diluted starting material is simultaneously separated andthe product harvested via one MF and one UF membrane workingsimultaneously.

What is claimed is:
 1. A method to isolate carbohydrates from a viscousplant material, said process comprising: contacting the viscous plantmaterial with a diluent in an amount sufficient to reduce the viscosityof the viscous plant material and form a continuous stream of dilutedplant material, wherein the diluent is contained in a separated vesselfrom the viscous plant material; flowing the diluted plants materialinto a recirculation loop of a first cross-flow filter apparatus;diafiltering the diluted plant material with sufficient diafiltrationbuffer retained in a buffer reservoir so as to recover the desired yieldof the carbohydrates by passing said carbohydrates into the firstpermeate fluid; flowing the first permeate fluid containing thecarbohydrates to an end product vessel; flowing out the first retentatesolution from the recirculating liquid of the first cross-flow filterinto a second cross-flow filter unit, wherein the flow rate of the firstretentate solution is at the same flow rate as the diluted plantmaterial being fed into the recirculation loop of the first cross-flowfilter apparatus; diafiltering the flow of retentate into the secondcross-flow filter unit with sufficient diafiltration buffer so as torecover the desired yield of the carbohydrates by passing saidcarbohydrates into the second permeate fluid; flowing the secondpermeate fluid containing the target substance to the end productvessel; concentrating the first and second retentate fluid by flowingsame to a third cross-flow filter apparatus communicatively connectedwith the second cross-flow filter unit, wherein the volume of the thirdretentate fluid is reduced to the approximate volume of the undilutedplant material or less thereby forming a waste stream for further use;recirculating the third permeate fluid back to the diluent vessel forreuse; concentrating the first and second permeate fluid by flowing sameto a fourth cross-flow filter apparatus communicatively connected to theend product vessel wherein the carbohydrates are concentrated anddiafiltration buffer is removed in fourth permeate stream andrecirculated for reuse.
 2. The method of claim 1, wherein the amount ofbuffer introduced into the buffering vessel is conserved and availablefor reuse.
 3. The method of claim 1, wherein the diluent is returned todiluent vessel simultaneously with the concentration of thecarbohydrates and the return of the buffer to the buffer reservoir. 4.The method of claim 1, wherein the cross-flow filters comprises: amultilaminate array of sheet members of generally rectangular andgenerally planar shape with main top and bottom surfaces, wherein thesheet members include in sequence in the array a first retentate sheet,a first filter sheet, a permeate sheet, and second filter sheet, and asecond retentate sheet, wherein each of the sheet members in the arrayhas at least one inlet basin opening at one end thereof, and at leastone outlet base opening at an opposite end thereof, with at least onepermeate passage opening at longitudinal side margin portions of thesheet members; each of the first and second retentate sheets having atleast one channel opening therein, wherein each channel opening extendslongitudinally between the inlet and outlet basin openings of the sheetsin the array and is open through the entire thickness of the retentatesheet, and with each of the first and second retentate sheets beingbonded to an adjacent filter sheet about peripheral and side portionsthereof, with their basin openings and permeate passage openings andregister with one another, and arranged to permit flow of filtratethrough the channel openings of the retentate sheet between the inletand outlet basin openings to permit permeate flow through the filtersheet to the permeate sheet to the permeate passage openings; and thecross-flow filters comprising a unitary article of inter-bonded sheetmembers.
 5. The method of claim 1, wherein the plant material is soy,potato, or tobacco.
 6. The method of claim 4, wherein the cross-flowfiltration module comprises channel height and length of the retentatesheet for optimal production yield.
 7. A system comprising: a firstreservoir constructed and arranged for holding a diluent solution, andfor selectively flowing liquid into and out of said first reservoir; asecond reservoir constructed and arranged for holding a startingmaterial, and for selectively flowing liquid into and out of said secondreservoir, a first cross-flow filtration apparatus for separatingliquids into permeate and retentate streams, provided with means forflowing liquid in from the first and second reservoir and permeate andretentate streams out of said first cross-flow filtration apparatus; asecond cross-flow filtration apparatus for receiving retentate from thefirst cross-flow filtration apparatus and separating liquids intopermeate and retentate streams, provided with means for flowing liquidin and permeate and retentate streams out of said second cross-flowfiltration apparatus; a third reservoir constructed and arranged forholding a buffer, and for selectively flowing liquid into the first andsecond cross-flow filtration apparatus and out of said third reservoirand; a third cross-flow filtration apparatus for separating liquids intopermeate and retentate streams, provided with means for flowing liquidin and permeate and retentate streams out of said third cross-flowfiltration apparatus; a product reservoir constructed and arranged forholding the isolated product received as permeate from the first andsecond cross-flow filtration apparatus, and for selectively flowingliquid into and out of the product reservoir.